Evaluation of kit-based loop-mediated isothermal amplification (TB-LAMP) assay in the diagnosis of tubercular lymphadenitis

Background. The rapid and accurate diagnosis of tubercular lymphadenitis remains a challenging task today. The World Health Organization (WHO) endorsed the LoopAMP MTBC kit (TB-LAMP) as a replacement for sputum smear microscopy in the diagnosis of pulmonary tuberculosis (PTB). However, no prospective diagnostic accuracy study of TB-LAMP for tubercular lymphadenitis in adults has been performed yet. The current study evaluated the diagnostic performance of TB-LAMP in tubercular lymphadenitis (LNTB). Methods. In a prospective observational study conducted at a tertiary care hospital in India, 90 subjects (age >18 years) suspected of LNTB were recruited consecutively and followed up for 6 months between January 2019 and December 2020. Samples were processed for microscopy, culture, GeneXpert, histopathology and TB-LAMP. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TB-LAMP against the composite reference standard (CRS) and culture were determined. Results. TB-LAMP showed a sensitivity of 83.78 % (95 % CI, 73.76–90.47) and a specificity of 81.25 % (95 % CI, 56.99–93.41), respectively, against the CRS. The PPV and NPV were 95.38 % (95 % CI, 87.29–98.42) and 52.00 % (95 % CI, 33.50–69.97), respectively. TB-LAMP showed a sensitivity of 88.89 % (95 % CI, 71.94–96.15) and a specificity of 36.17 % (95 % CI, 23.97–50.46), respectively, against culture. The PPV and NPV were 44.44 % (95 % CI, 32–57.62) and 85 % (95 % CI, 63.96–94.76), respectively. Conclusion. TB-LAMP can be used instead of conventional microscopy for the diagnosis of TB in lymph node specimens at primary healthcare centres. It provides rapid and cost-effective diagnosis of LNTB in resource-limited settings due to good sensitivity and NPV.


INTRODUCTION
Tuberculosis (TB) has plagued humanity for a long time.In 1993, the World Health Organization (WHO) declared TB a global health emergency [1].There were an estimated 10 million new cases of TB worldwide in 2019, with an estimated 1.4 million deaths.India had the largest share (26 %) of the estimated new cases, with 2.64 million new cases and 0.45 million deaths from TB in 2019.Previous studies have estimated extrapulmonary TB (EPTB) to constitute 15-20 % of the total TB cases and up to 50 % of HIV-TB coinfections [2].Tubercular lymphadenitis (LNTB) constitutes ~35 % of total EPTB cases [3].As per National TB Elimination Programme (NTEP) data [4], the incidence of LNTB in India was 30.8 per 100 000 population in 2013.LNTB is a local manifestation of underlying systemic disease [5].Thus, prompt diagnosis with timely initiation of appropriate therapy is essential.
For the past few decades, acid-fast bacillus (AFB) smear microscopy has been the mainstay of diagnosis of pulmonary tuberculosis (PTB), especially in resource-poor settings, due to its low cost and rapid results [6].However, it is observerdependent and has poor sensitivity.TB diagnosis by culture requires a long incubation time of 6-8 weeks.Nucleic acid amplification tests (NAATs) such as polymerase chain reaction (PCR) aid in the rapid, specific and sensitive diagnosis of LNTB but require technical expertise and expensive equipment.The GeneXpert (Cepheid, Sunnyvale, CA, USA) assay is limited by cost and dependence on sophisticated equipment/software.
The search for a true point-of-care test to aid the rapid, accurate and low-cost diagnosis of EPTB in a peripheral setting with limited resources is still ongoing.One of the promising candidates to have emerged so far is LoopAMP (LAMP), another easy-to-use, isothermal NAAT that relies on strand displacement DNA synthesis performed by DNA polymerase with high strand displacement activity in an autocycled reaction using two inner and two outer primers that recognize a total of six distinct sequences on the target DNA [7,8].It provides visible results that can be read easily and performed even in peripheral centres and has already been used to detect severe acute respiratory syndrome (SARS), malaria, influenza and African trypanosomiasis.One such test is the LoopAMP MTBC kit (TB-LAMP; Eiken Chemical Company, Tokyo, Japan), a commercial kit employing TB-LAMP technology to detect Mycobacterium tuberculosis complex (MTBC) rapidly in samples.The WHO has recommended TB-LAMP as a replacement test for sputum smear microscopy to diagnose PTB in adults with clinical features suggestive of PTB and as a follow-on test to smear microscopy in adults with clinical features suggestive of PTB, especially in sputum smear-negative specimens with a high index of clinical suspicion [6].No guidelines have been formulated yet for the use of TB-LAMP in the diagnosis of LNTB.
Culture, although still considered the gold standard for LNTB diagnosis, has been termed 'an imperfect reference standard' [9].In the systematic review performed by Kohli et al. in 2018 evaluating the GeneXpert assay in EPTB diagnosis, they found that culture did not perform well as the reference standard for LNTB [10].Imperfect reference standards may lead to the wrong classification of subjects as cases and non-cases in diagnostic accuracy studies.The concept of a composite reference standard (CRS), comprising the results of several tests such as AFB smear, culture, histopathology/cytopathology, clinical evaluation and treatment response, was introduced to solve this issue [11,12].Vadwai et al. also used a CRS in their diagnostic accuracy study of GeneXpert in EPTB samples [13].However, a CRS might have reduced specificity, leading to the misclassification of non-cases as cases.This would lead to wrong estimation of the sensitivity of the diagnostic test being evaluated, which would be lower than the actual sensitivity.Thus, a study evaluating TB-LAMP with culture and CRS, both used as reference standards, would be most appropriate to obtain a credible range of sensitivity and specificity [9].
We conducted a prospective observational study to evaluate the diagnostic accuracy of TB-LAMP for detecting LNTB, compared with CRS and culture, and assess the interrater reliability between TB-LAMP and GeneXpert.

Study setting
In this prospective observational diagnostic accuracy study, 90 patients (aged >18 years) were recruited consecutively from outpatient and inpatient settings, including the Infectious Diseases clinic and the medical ward.The treating physician advised during the routine investigations, after which the patients were screened and recruited after obtaining written informed consent.The study was conducted between January 2019 and December 2020 at All India Institute of Medical Sciences (AIIMS) New Delhi, India.A 6 month post-recruitment follow-up was conducted to assess the clinical response to anti-tubercular therapy (ATT) and the evolution of symptoms (Fig. 1).

Sample size
We considered the sensitivity and specificity of AFB smear to be 76.47 and 100 %, respectively, while those of culture were 88.23 and 100 %, respectively, according to the reference study by Mittal et al. [14].We also considered the sensitivity and specificity of GeneXpert in LNTB to be 81.20 and 99.10 %, respectively, according to the systematic review by Denkinger et al. [15].At 95 % confidence interval and a margin of error of 7 %, and anticipating an increase in the sensitivity and specificity of CRS to 90 and 98 %, respectively (since CRS comprises AFB smear, culture and GeneXpert), the required number of cases and non-cases aweree 71 and 16, respectively.

Inclusion criteria
Patients (>18 years of age) with clinical features compatible with tubercular lymphadenitis (LNTB) as per Index-TB guidelines [16], i.e. fever >7 days, X-ray/CT showing enlarged lymph nodes suggestive of TB, tender/non-tender enlarged lymph nodes, anorexia, weight loss, cough, shortness of breath, night sweats, dull or colicky pain, who were not on anti-tubercular therapy, were included.

Exclusion criteria
Participants were excluded from the study if they refused to provide written informed consent, drug-resistant TB was detected or fine needle aspiration cytology (FNAC)/biopsy samples could not be obtained.

Procedures History and clinical examination
Patients attending the outpatient and inpatient settings were screened for symptoms compatible with LNTB, as mentioned in the inclusion criteria.History of symptom onset and duration and comorbidities, including diabetes mellitus, hypertension, HIV disease, previous history of TB, was taken.General physical examination of the enrolled study participants was performed in medical outpatient and inpatient settings, followed by a systemic examination (Table 1).

Culture
The decontaminated sample was used for inoculation in automated BACTEC MGIT 960 for liquid culture, following the standard protocol [17].

GeneXpert
The GeneXpert assay was performed according to the standard protocol [18].

TB-LAMP
The LoopAMP MTBC detection kit was used.First, 60 µl of the homogenized sample was transferred to a heating tube followed by heat inactivation at 95 °C for 10 min.The ultrarapid extraction (PURE) adsorbent tube was connected directly to the heating tube, and DNA extraction was completed.The PURE tube was assessed visually for the presence or absence of fluorescence from the reaction tube using UV light, after DNA amplification at 67 °C for 40 min, to determine the result according to the manufacturer's instructions [19].

Composite reference standard (CRS) and follow-up
Based on the clinical features, the results of histo-/cytopathological examination, AFB smear, MGIT culture, imaging or response to ATT, a CRS was devised that was similar to the one used by Vadwai et al. [13], and the participants were divided as follows.• Confirmed TB: culture-/smear-positive with clinical features suggestive of TB.
• Probable TB: both culture and smear-negative, but: • GeneXpert-positive or • Histopathological examination or imaging compatible with TB along with clinical response to ATT present.• Non-TB: culture and all other tests (GeneXpert, AFB staining, histopathology, imaging) for TB are negative, and the patient did not receive/respond to ATT.
For the final analysis, the confirmed and probable CRS categories were considered to be TB cases, while the non-TB CRS category was considered to be non-cases.

Statistical analysis
The data were summarized and analysed using STATA version 14 software (StataCorp LLC, USA) to describe the patient's demographics, clinical features, and examination and laboratory findings.Data were tested for normality using the Kolmogorov-Smirnov test.Data were expressed as mean+/−sd for normal distribution, median (IQR) or number and percentage as appropriate.
Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and likelihood ratio were calculated to assess the diagnostic accuracy of TB-LAMP against CRS and MGIT culture; 95 % confidence intervals were calculated using the Wilson score interval.Agreement between TB-LAMP and GeneXpert in LNTB was calculated using Cohen's kappa statistic.A P-value <0.05 was considered statistically significant.

RESULTS
Ninety participants with lymphadenopathy meeting the inclusion criteria were enrolled in the study, and a month follow-up was performed after enrolment, as per the study design.
There were 16 non-cases and 74 cases.Out of the 74 cases, 37 were in the confirmed TB and 37 in the probable TB category.
Out of the 16 non-TB subjects, 13 had a culture-negative report.A culture report was unavailable for the remaining three non-TB cases, and they were classified as non-TB cases based on the absence of response to ATT.All of the 16 non-TB subjects had a negative AFB smear.
Out of the 37 confirmed TB cases, 27 were identified based on a positive culture report.Out of the 27 TB cases with positive culture reports, only 12 were AFB stain-positive.Of the remaining 10 confirmed TB cases identified based on positive AFB staining, 7 had a negative culture report, and 3 did not have a culture report.Of the 37 probable TB cases, 27 had both negative culture and AFB smear reports, but a positive GeneXpert report and clinical response to ATT.The remaining 10 probable TB cases did not have a culture report and had a negative AFB smear, but had a positive GeneXpert report and a clinical response to ATT (Fig. 1).

Agreement between GeneXpert and TB-LAMP
The kappa statistic for agreement between TB-LAMP and GeneXpert was 0.44 (95 % CI, 0.23-0.65),which signifies a moderate agreement between the results of GeneXpert and TB-LAMP, as shown in Table 3.

DISCUSSION
We conducted a prospective observational study including 90 study participants with a clinical suspicion of LNTB, out of which 74 were cases and 16 were non-cases.The observation of a higher specificity of TB-LAMP compared to CRS instead of culture can be explained by the low sensitivity of culture in LNTB.Further, the decontamination practices using NALC-NaOH can further decrease viable bacilli in LNTB, a paucibacillary form of TB [16].False-positive TB-LAMP results can also be due to detection of dead bacilli and exposure to high humidity leading to non-specific false-positives, reducing the specificity of TB-LAMP.
In our study, the culture positivity rate was 36.40 % (27 positive reports out of 74 samples in TB cases).All 16 participants did not have a valid culture report due to an inadequate sample having been obtained.In comparison, the culture positivity rate was 15 % in the study by Sharma et al. [20], 15 % in the study by Mishra et al. [21] and 60 % in the study by Mittal et al. [14].Culture showed a sensitivity of 50 % (95 % CI, 37.11-62.89)and a specificity of 100 % (95 % CI, 77.19-100), respectively, against CRS.The PPV and NPV were 100 % (95 % CI, 87.54-100) and 32.5 % (95 % CI, 20.08-47.98),respectively.Similar values were reported in the studies by Vadwai et al. [13] and Mittal et al. [14].The low sensitivity of culture can be attributed to the paucibacillary nature of LNTB and the decontamination techniques used for processing samples, which might further decrease the viable bacilli number, as shown by Sharma et al. in their study [16].Wide-ranging values for culture sensitivity in the various studies and the low sensitivity in most studies have cast doubt on the use of culture as the sole reference standard in evaluating diagnostic tests in paucibacillary TB such as LNTB.
The level of statistical agreement between the results of TB-LAMP and GeneXpert was moderate; Cohen's kappa statistic of 0.44 (95 % CI, 0.23-0.65).In our study, GeneXpert had a sensitivity of 94.59 % (95 % CI, 86.91-97.88)and a specificity of 100 % (95 % CI, 80.64-100) against CRS, respectively.Thus, GeneXpert was superior to TB-LAMP for detecting LNTB with the additional benefit of rifampicin resistance detection and estimation of bacillary load.The discrepancy between GeneXpert and TB-LAMP results could be due to the presence of inhibitors of DNA replication affecting GeneXpert, but not TB-LAMP.Simultaneously, the different DNA extraction kit provided by the manufacturer may be the other cause of the discrepancy between these diagnostics modalities.
The major limit of the present study is a slightly higher but acceptable margin of error of 7 % that had to be employed in our study with its small sample size due to logistical and cost constraints.Multidrug-resistant TB and rifampicin-resistant cases on GeneXpert were excluded from our study.It should also not be possible to replace rapid molecular tests with higher sensitivity for MTBC along with DR-TB detection.The LAMP assay used in the study is a commercial assay for the detection of MTBC.The difference in our results compared to previous studies can also be explained by the difference in genes targeted by the LAMP assays used, as shown in Table 4 [18,22,23].While the TB-LAMP kit used in our study targets IS6110 and gyrB, the in-house LAMP assay used by Sharma et al. [16] targeted IS6110 and MPB64.Combinations of primers targeting various genes should be further explored to obtain a test with the best diagnostic accuracy.The study was conducted in a tertiary care hospital, while TB-LAMP is intended to be used for the diagnosis of MTBC as a point-of-care test in peripheral or rural settings instead of conventional microscopy, where results may be influenced by lower bacillary load and inappropriate sample collection and processing in the case of EPTB specimens.

Conclusion
Given its sensitivity of 84-89 % and specificity of 36-81 % compared to culture and CRS and its relative ease of use in peripheral settings due to there being no requirement for thermal cycling or sophisticated equipment, in addition to its shorter turnaround time, TB-LAMP may play a role in the diagnosis of MTBC as a point-of-care test for rapid and cost-effective diagnosis of LNTB in resource-limited, peripheral settings.However, TB-LAMP does not give any information regarding drug resistance or bacillary load, which is a drawback in comparison to GeneXpert, which can detect rifampicin resistance by detecting mutations in the rpoB gene.Thus, it can be used as the initial diagnostic as well as a follow-up test instead of smear microscopy, followed by referral to higher centres for mycobacterial culture and GeneXpert.Combinations of primers targeting various genes should be explored further to obtain a test with the best diagnostic accuracy.The Microbiology Society is a membership charity and not-for-profit publisher.
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Find out more and submit your article at microbiologyresearch.org However, where a culture report was available, it was considered as a case outright irrespective of AFB stain.In the 10 confirmed CRS group (out of total 37 confirmed CRS group) who either did not have a culture report or had a negative culture report, AFB stain was considered.Response to treatment was not used in the classification of these 10 patients as cases, although we have follow-up data for all these patients which showed response in our outpatient clinic.

2.
One important analysis to do is with sputum smear microscopy only.Potentially that will have a higher impact as the WHO guidance is to potentially replace sputum smear microscopy in countries or places that solely rely on smear microscopy.This has not been analyzed and discussed in the paper.The intended use of this technology needs to be indicated.
Ans:Since a positive AFB smear microscopy was used as a criteria for the classification of only 10 patients as a confirmed case, it wasn't discussed in the paper and more focus was on the analysis and comparison with CRS and culture used as a gold standard for diagnosis.When AFB smear microscopy was compared to CRS as a gold standard, the sensitivity and specificity were 29.73% (20.53%, 40.93%) and 100% (80.64%, 100%), respectively.The 100% specificity was due to inclusion of AFB smear positivity being one of the criteria for direct classification of the patient as a case, if culture report is either negative or unavailable.When AFB smear microscopy was compared to MGIT culture as a gold standard, the sensitivity and specificity were 44.44% (27.59%, 62.69%) and 85.11% (72.31%, 92.59%), respectively.
The Cohen's kappa correlation coefficient for agreement between TB-LAMP and AFB smear microscopy was 0.15 (0.02-0.28),only slight agreement.The poor agreement can be explained by the poor sensitivity of AFB smear which led to 42 AFB negative TB cases being detected by TB-LAMP.TB-LAMP however failed to detect 2 AFB smear positive cases which were both GeneXpert positive and one was MGIT culture positive too.Thus, the use of TB-LAMP as a replacement for AFB smear microscopy is to be used as a sensitive test which would help peripheral facilities to refer likely TB cases for treatment and evaluation to higher center under national programmes.

3.
The authors should also mention the generalizability aspect in their results.The patients were taken from a tertiary care Centre.The deployment of this technology is at POC level, where the bacillary load might be even less and difficult to diagnose the disease.This should be mentioned in their discussion section as one of the potential concerns.
Ans:Yes.This point is well-taken and has been incorporated in the discussion.However, due to the lower limit of detection of TB-LAMP, the low bacillary load at a peripheral POC level due to technical or biological reasons, makes TB-LAMP a better alternative to AFB microscopy.

4.
Reasons for why few cases were GX+ Lamp-and vice versa should be discussed.In-depth knowledge of molecular markers, technical performance of different techniques and rationales for comparison between the two tests are lacking.
Ans:Yes, the point is well taken.We have already discussed about this in discussion section.This may be due to some inhibitor present and very low bacterial load in the specimens.

Reviewer 2
Comments to Author: It is observed that biopsy was done for 19 subjects and hence histopathology reports should have been available.Correlation of LAMP with histopathology /FNAC pathology findings have not been included in the paper.What was the correlation?Were positive results detected for biopsies with other diagnosis especially malignancy?What could have been the impact on such positives on original disease?This is important since LAMP is being recommended at peripheral facilities without adequate infrastructure.
One positive result on TB-LAMP was detected for biopsy histopathologically reported as a case of Hodgkin lymphoma with negative AFB stain and MGIT culture.GeneXpert was positive in the same patient and he was followed up and showed improvement both clinically and biochemically (improvement in inflammatory markers) on treatment.He was already under medical oncology follow-up and treatment for Hodgkin's lymphoma and had likely contracted tuberculosis which was detected during evaluation of persistent fever with cervical lymphadenopathy and elevated inflammatory markers.Comments: Thank you for submitting your manuscript for publication in Access Microbiology.It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology.However, based on the comments received, a minor amendment of this manuscript will be required before a decision can be made on its publication.I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments.Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers' comments.Anonymous.
© 2023 Allen D. This an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Reviewer 2 recommendation and comments https
://doi.org/10.1099/acmi.0.000665.v1.4 © 2023 Chandrasekaran S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.SRM institute for Medical Sciences, Cl.Microbiology & Serology, Vadapalani, Chennai, INDIA https://orcid.org/0000-0002-0821-6768Datereportreceived:24September2023 Recommendation: Accept Comments: Methodology , analysis are satisfactory.It is observed that biopsy was done for 19 subjects and hence histopathology reports should have been available.Correlation of LAMP with histopathology /FNAC pathology findings have not bee included in the paper.What was the correlation?Were positive results detected for biopsies with other diagnosis esp malignancy?What could have been the impact on such positives on original disease?This is important since LMP is being recommended at periphaeral facilities without adequate infrastructure.Please rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000665.v1.3 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.